495 1
J . Am. Chem. SOC.1984, 106, 4951-4956
Resonance Raman Study of the p-Oxo-Bridged Binuclear Iron Center in Oxyhemerythrin Andrew K. Shiemke, Thomas M. Loehr, and Joann Sanders-Loehr* Contribution from the Department of Chemistry and Biochemical Sciences, Oregon Graduate Center, Beaverton, Oregon 97006. Received February 29, 1984
Abstract: Oxyhemerythrin, the respiratory protein of the marine sipunculid Phuscolopsis gouldii, was investigated by resonance Raman spectroscopy. The presence of a p-oxo-bridged binuclear iron center in oxyhemerythrin was established by finding v,(Fe-0-Fe) at 486 cm-I with ultraviolet excitation. In H2I80solvent, the 486-cm-' vibrational mode shifts to ~ 4 7 cm-I, 5 a shift similar in magnitude to that observed for other p-oxo-bridged Fe(II1) proteins, such as several methemerythrins and ribonucleotidereductase. A study of the enhancement profile of the v,(Fe-@Fe) mode in oxyhemerythrinindicates that resonance occurs by coupling to the -360-nm transition and serves to assign this band to an 02- Fe(II1) charge-transfer process. By contrast, although the v,(Fe-O-Fe) mode in azidomethemerythrin at 507 cm-l is also strongly enhanced in the UV, this mode is observable with visible-light excitation. Its enhancement profile is complex, showing apparent maxima at 4 2 5 , 430, and --Tris (pD 8.55). The D 2 0 buffer exchange was accomplished in either of two different ways: (1) protein was dialyzed against the D 2 0 buffer for =2 days; (2) concentrated protein (-6 mM) was applied to a Sephadex G-25 column (2.5-mL column volume) which had been saturated with D 2 0 buffer and was then eluted with D 2 0 buffer. The second method was much faster, being complete in =30 min, though both methods gave identical results. Azidomethemerythrin in D 2 0 buffer was prepared by adding solid NaN3 to oxyhemerythrin in D 2 0 buffer, to give a final azide concentration of 0.01 M. Spectroscopy. Resonance Raman spectra were collected on a computer-interfaced Jarrell-Ash spectrophotometer21equipped with Spectra-Physics 164-05 (Ar) and 164-01 (Kr) ion lasers, an RCA C31034 photomultiplier tube, and an ORTEC Model 9302 amplifier/discriminator. Both lasers are equipped with ultra-high-field magnets to enhance ultraviolet output. Typical sample concentrations were 1-1.5 mM in hemerythrin monomer. Sample decomposition was minimized through use of a flow cell. A reservoir of 2-3 mL of protein solution was placed in a constant-temperature bath maintained at 3-5 O C . The sample was pumped through 0.8-mm i.d. tygon tubing and a 1.6-mm 0.d. quartz capillary tube (situated transverse to the incident laser beam) in a recirculating system with a flow rate of 2 1 . 5 mL/min. In all cases a 9O0-scattering geometry was used, and multiple scans were collected to enhance the signal-to-noise ratio. Sample integrity was monitored by obtaining optical spectra (Cary 16) both before and after exposure to the laser beam. Only upon prolonged exposure of oxyhemerythrin to UV laser light was there any appreciable sample degradation; however, even these samples typically retained >80% activity as measured by the ASm/A326 ratio, using extinction coefficients reported by Dunn et ai.' For ( u I = 981 cm-') the resonance Raman enhancement profiles, 0.3 M was used as the internal intensity standard. Corrections for self-absorption were made22 but found to be insignificant compared to the uncertainty inherent in the measurement. Results and Discussion F e F e Vibrations. The resonance R a m a n spectrum of oxyhemerythrin obtained with 363.8-nm excitation is shown in Figure 1. In H,I60, t h e protein shows a strongly enhanced feature a t 3 W h e n the protein 486 cm-' a n d a weaker feature a t ~ 7 5 cm-l.
(17) Kurtz, D. M.; Shriver, D. F.; Klotz, I. M. Coord. Chem. Reu. 1977, 24, 145-178.
(18) York, J. L.; Bearden, A. J. Biochemistry 1970, 9, 4549-4554. (19) Clark, P. E.; Webb, J. Biochemistry 1981, 20, 4628-4632. (20) Klotz, I. M.; Klotz, T. A.; Fiess, H. A. Arch. Biochem. Biophys. 1957, 68, 284-299.
(21) Loehr, T. M.; Keyes, W. E.; Pincus, P. A. Anal. Biochem. 1979, 96, 456-463. (22) (a) Dum, J. B. R.; Shriver, D. F. Appl. Specrrosc. 1974, 28, 319-323. (b) Strekas, T. C.; Adams, D. H.; Packer, A,; Spiro, T. G . Ibid. 1974, 28, 324-327,
Binuclear Iron Center in Oxyhemerythrin 7
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Figure 2. Resonance Raman enhancement profiles for oxyhemerythrin. (Upper) Electronic absorption spectrum of oxyhemerythrin. (Middle) Enhancement profile for the symmetric F e U F e vibration at 486 cm-l. (Lower) Enhancement profiles for the vibrations of the bound peroxide: 0-0 stretch at 844 cm-l; Fe-O2 stretch at 503 cm-l. Enhancement measured as the height of the Raman peak relative to the height of v I of 0.3 M SO-: at 981 cm-' and normalized to the ~ ( F e - 0 ~height ) obtained with 530.9-nm excitation. Table I. Vibrational Frequencies (cm-') for the Stretching and Deformation Modes of the Fe-0-Fe Cluster in Hemerythrins and Model ComDounds
v,(Fe-0-Fe)" sample hemerythrin
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720 n.0. n.0. 475 -753 486 733 292 286 493 768 azidomet 501 -285 742 -290 498 780 514 thiocyanatomet n.d. -715 n.0. 496 -750 510 met n.d. hydroxomet 780 -750 n.0. 508 n.d. 782 n.d. n.0. cyanatomet n.d. 509 493' 780 n.d. n.0. n.d. 512 499' cyanomet model compound 528 511 751 721 283 269 Fe,O(CH,COO),-. (HB(PZY,)~~ [Fez0 (phen) 4395 390 827 788 no. n.0. (H20)214+C [ Fe20C16]2-d 458 440 870 826 203 198 a Values obtained by Raman spectroscopy (n.d. = not determined; values for the methemerythrins n.0. = not observed). The v,(Fe-O-Fe) in H 2 0 are similar ( f l cm-I) to those reported previ0us1y.l~ bReference 27. 'Reference 29. dReference 26. 'Values for hemerythrin obtained by Raman spectroscopy. Values for model compounds obtained by I R spectroscopy. Uncertainty in frequencies (indicated by -) due to the interference of other protein peaks. 'Reference 13. OXY
is prepared in H2180,these two peaks shift to lower energy by 11 and 33 cm-I, respectively (Figure 1 and Table I). The isotope dependence identifies these vibrations as Fe-0 modes and the exchangeability with solvent oxygen implies that they are associated with an Fe-0-Fe moiety in oxyhemerythrin. The 486-cm-' peak can be assigned to the symmetric stretching vibration on the basis of its intensity, frequency, depolarization ratio, and the magnitude of its 0-isotope shift (see below). The symmetric Fe-0-Fe mode has been observed previously in a number of methemerythrin species by using visible-light excitation, but never
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Figure 3. Resonance Raman enhancement profiles for azidomethemerythrin. (Upper) Electronic spectrum of azidomethemerythrin. (Middle) Enhancement profile for the symmetric Fe-0-Fe vibration at 507 cm-'. (Lower) Enhancement profiles for the FeN(azide) stretch at 375 cm-l, N-N(azide) stretch at 2048 cm-I, and Fe-0-Fe deformation at 292 cm-l. Enhancement measured as in Figure 3 but normalized to the v(Fe-N,) height obtained with 496.5-nm excitation.
in ~xyhernerythrin.~~~" The failure to detect this vibrational mode of oxyhemerythrin was due to the fact that, in contrast to the methemerythrins, it is only enhanced with near-UV excitation (Figure 2). Thus, the present findings provide the first direct evidence for the existence of a p-oxo bridge in the binuclear iron site of oxyhemerythrin. Our ability to exchange the p-oxo-bridge oxygen of oxyhemerythrin is also a new result. In earlier experiments Kurtz et aI.l7 and Freier et al.l3 were unable to observe '*O-exchange during the conversion of deoxy to oxyhemerythrin. In their work, crystals of oxyhemerythrin were dissolved in H2180buffer, reduced with dithionite, and then immediately r e o ~ y g e n a t e d . ' ~The . ~ ~only obvious procedural differences are that our deoxyhemerythrin was prepared by dithionite reduction of (thiocyanat0)methemerythrin and that the deoxyhemerythrin (with or without crystallization) was equilibrated in H2180buffer for 2 days prior to oxygenation. The longer period of exposure of deoxyhemerythrin to H2I80is the most likely explanation for the exchange observed in our experiments. Alternatively, it is possible that we achieved a conformation of deoxyhemerythrin that is more susceptible to bridge exchange by having performed the reduction on the thiocyanato (as opposed to the oxy) form of the protein. Kinetic studies have shown that (thiocyanat0)rnethemerythrin undergoes more facile reduction with dithionite than other forms of meth e m e r ~ t h r i n . ~Moreover, ~ the mixed valence Fe(II),Fe(III) semi-methemerythrins are believed to exist in several different conforma ti0ns.2~ The u,(Fe-O-Fe) assignment in oxyhemerythrin is compatible with analogous vibrations which have been observed in the Raman spectra of other oxo-bridged c o m p o u n d ~ . 2 ~Systems -~~ that have (23) Olivas, E.; deWaal, D. J. A.; Wilkins, R. G. J . Inorg.Biochem. 1979, 11, 205-212. (24) Bradic, Z.; Harrington, P. C.; Wilkins, R. G.; Yoneda, G. Biochemisrry 1980, 19, 4149-4155. (25) Burke, J. M.; Kincaid, J. R.; Spiro, T. G. J . Am. Chem. Soc. 1978, 100, 6077-6083.
4954 J . Am. Chem. SOC.,Vol. 106, No. 17, 1984 been shown to contain a nonlinear Fe-0-Fe structural unit and give rise to a resonance-enhanced symmetric Fe-O-Fe vibration near 500 cm-l are ribonucleotide reductase,28 F e 2 0 4+,29 and all forms of (CH,COO),( H B ( P Z ) ~ ) , ,[~Fe20(phen)4] ~ metherner~thrin'~(other than sulfidomethemerythrin). The corresponding isotope shifts for v,(Fe-0-Fe) with I8O are -15 cm-I for ribonucleotide reductase,28-5 to -18 cm-' for the model compounds (Table I), and -10 to -16 cm-I for the methemerythrins (Table I). The -1 1-cm-' shift for oxyhemerythrin is well within this range. Furthermore, all of these systems appear to show maximal resonance enhancement with UV excitation. Although the v,(Fe-0-Fe) mode in methemerythrins was originally observed in resonance with visible electronic transitions,7J3 we have now found additional enhancement with UV excitation for the N3- (Figure 3, middle), CN-, CNO-, SCN-, OH-, CI-, ClO,-, and met (anion-free) forms of the protein. Thus, the 486-cm-' peak in the Raman spectrum of oxyhemerythrin shows the characteristics expected for the symmetric stretching vibration of a bent Fe-0-Fe moiety. The additional oxygen-isotope sensitive vibrational mode at -753 cm-I in oxyhemerythrin can be assigned to the asymmetric Fe-0-Fe vibration on the basis of its frequency, its low intensity relative to v,(Fe-O-Fe), and its isotope shift. An analogous oxygen isotope-sensitive peak was observed in every form of methemerythrin we investigated (Table I). In all cases, the v,,(Fe-O-Fe) was maximally enhanced with UV excitation. For oxyhemerythrin and azidomethemerythrin, the UV enhancement profiles of uas(Fe-0-Fe) appeared to parallel those of v,(Fe-0-Fe). The intensity of the asymmetric vibration was difficult to quantitate due to the presence of an additional spectral feature at =757 cm-I which becomes apparent in H2I80(Figure 1). Since this feature was also discernible in all of the methemerythrin samples studied in both H2I60and H2I80and is not attributable to the Raman buffer at this concentration, it is most likely due to a protein moiety such as t r y p t ~ p h a n . Although ~~ accurate depolarization ratios could not be obtained for the 750-780-cm-' feature, it appears to be depolarized as expected for an asymmetric vibration. The 750-780-cm-' frequencies and the -33 to -38 cm-I isotope shifts of the asymmetric bridge modes in hemerythrins are comparable to the behavior of p-oxo-bridged model complexes (Table I). The closest agreement is with the pyrazolylborate which has a similar small Fe-0-Fe bridging angle of 123.5' compared to 135O in azidomethemerythrin.31 The bond angles in the other two model compounds are -155' and, thus, are expected to have their asymmetric vibrational modes at higher energy?, In model compounds u,(FeO-Fe) is generally observed by IR spectroscopy. The phenanthroline and chloride complexes also give weak Raman bands at corresponding frequencies, but only the protein shows clear-cut resonance enhancement of this vibrational mode. It has been suggested that the protein band is a V, 6 combination band as in the pyrazolylborate complex.27 This seems unlikely since the protein peak at -750 cm-I exhibits a much greater isotope shift than would be expected for a combination band from a Aus of -1 1 to -16 and a A6 of -6 cm-' (see below). A third H2180-sensitivevibration has been detected in several methemerythrins. In azidomethemerythrin, the peak at 292 cm-' shifts to 286 cm-I in the I80-substituted protein (Figure 4). This vibrational mode had previously been assigned to v(Fehi~tidine).~.'~ However, it is more appropriately described as the deformation mode of the Fe-UFe moiety. Analogous vibrations
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(26) Solbrig, R. M.; Duff, L. L.; Shriver, D. F.; Klotz, I. M. J . Inorg. Biochem. 1982, 17, 69-74. (27) Armstrong, W. H.; Spool, A.; Papaefthymiou, G. C.; Frankel, R. B.; Lippard, S. J. J . Am. Chem. SOC.1984, 106, 3653-3661. (28) SjBberg, B.-M.;Loehr, T. M.; Sanders-Loehr, J. Biochemistry 1982, 21, 96-102. (29) Plowman, J. E.; Loehr, T. M.; Schauer, C. K.; Anderson, 0. P. Inorg. Chem., in press. (30) Lord, R. C.; Yu, N.-T. J . Mol. Biol. 1970, 50, 509-524. (31) Stenkamp, R. E.; Sieker, L. C.; Jensen, L. H. J . Am. Chem. SOC. 1984, 106, 618-622. (32) Wing, R. M.; Callahan, K. P. Inorg. Chem. 1969,8, 871-874.
Shiemke, Loehr, and Sanders-Loehr AZIDOMETHEMERYTHRIN '5
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-
have been detected in the Raman spectra of two of the oxo-bridged model compounds ( A = -5 to -14 cm-I in H2I80) and probably also in (thiocyanat0)methemerythrin (Table I). However, no 6(Fe-O-Fe) frequencies have yet been observed in either oxyhemerythrin or the hydroxo, cyanato, cyano, and ligand-free forms of methemerythrin. Enhancement P r o f h Exogenous Ligands. Analysis of Raman spectral intensities as a function of excitation wavelength gives information about the nature of the electronic transitions responsible for the vibronic coupling. The electronic spectra for oxyhemerythrin and azidomethemerythrin are shown in the upper panels of Figures 2 and 3, respectively. The lower panels illustrate enhancement profiles for the vibrations due to the exogenous ligands, 0:- and N3-. The data for the visible region are in good agreement with previously published profiles.'J3 The intensity of ~ ( F e - 0 ~of) oxyhemerythrin maximizes at 525 nm whereas the intensities of u(Fe-N,) and v(N-N) of azidomethemerythrin maximize at 490 and 480 nm, respectively. We have extended these profiles into the near-UV region of the electronic spectrum. The 0-0 stretching vibration at 844 cm-l in oxyhemerythrin steadily decreases in intensity with increasing frequency of the incident light, vanishing with near-UV excitation. This is not the case for the stretching modes due to Fe-N3 at 375 cm-' and N-N at 2048 cm-l in the azidomethemerythrin spectrum or Fe-0, at 503 cm-I in the oxyhemerythrin spectrum. All three of these vibrations show enhancement into the near-UV; the Fe-0, stretching mode appears to be coupled to the 360-nm transition in oxyhemerythrin, whereas the Fe-N, and N-N stretching frequencies appear to be coupled to the 325-nm transition in azidomethemerythrin. The enhancement profiles for v(Fe-O2) and u(Fe-N3) are in excellent agreement with the single-crystal spectroscopic studies by Solomon et a1.I5J3 These authors observed electronic transitions that were polarized perpendicular (I) to the Fe-Fe axis and assigned them to charge transfer resulting from end-on binding (33) Solomon, E. I.; Eickman, N . C.; Gay, R. R.; Penfield, K. W.; Himmelwright, R. S.; Loomis, L. D. In "Invertebrate Oxygen Binding"; Lamy, J., Lamy, J., Eds.; Marcel Dekker: New York, 1981; p 487.
J. Am. Chem. SOC.,Vol. 106, No. 17, 1984 4955
Binuclear Iron Center in Oxyhemerythrin of peroxide or azide to a single iron atom. The I-polarized 0 in oxyhemerythrin are pretransitions a t =50G and ~ 3 6 nm sumably responsible for the Raman enhancement maxima of u(Fe-OZ) at 525 and =360 nm. Similarly, the I-polarized transitions a t =490 and =325 nm in azidomethemerythrin are consistent with the observed Raman enhancement maximum of v(Fe-N,) at 490 nm and an additional maximum
